D2 dopamine receptors have been implicated in neuropsychiatric and neurologic disorders including schizophrenia, drug abuse, and Parkinson's disease. Acute activation of D2 dopamine receptors inhibits cyclic AMP accumulation;however, persistent activation of D2 dopamine receptors enhances subsequent drug-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylyl cyclase (AC) signaling occurs following persistent activation of several G?i/o-coupled receptors in vitro and in vivo. The overall objective of this research proposal is to elucidate the molecular mechanisms involved in heterologous sensitization of AC following persistent activation of D2-like dopamine receptors. Previous studies support a hypothesis that heterologous sensitization requires the activation of G?i/o subunits to induce sensitization through a G??-dependent mechanism. We hypothesize that G?? subunits lead to heterologous sensitization of individual AC isoforms through both direct and indirect mechanisms. The indirect mechanisms may involve protein-protein interactions as well as G?s. The general approach for these studies will be to express heterologously D2L dopamine receptors together with well characterized wild-type or mutant ACs (e.g. AC1, AC2, and AC5) for intact cell experiments in unique cellular backgrounds (i.e., G protein subunit deficient). This strategy takes advantage of recently discovered molecular and cellular tools to study G protein signaling as well as novel fluorescent technologies. The first specific aim will test the hypothesis that heterologous sensitization of select isoforms of AC involves G??-AC interactions and requires G?? subunit signaling. These studies will use a series of AC mutants, unique cellular models, small molecule inhibitors of G?? subunit signaling, and striatal neurons. The second specific aim will determine the roles and requirements for G protein subunits in modulating receptor-AC and AC-AC interactions. These experiments will use bimolecular fluorescence complementation (BiFC) to probe the specific role of G?? and G?s subunits in modulating basal and drug-induced protein-protein interactions in living cells. The third specific aim will identify and characterize the AC "sensitization interactome" using BiFC in a neuronal cell model. These studies will use BiFC to perform cDNA library screening to identify sensitization-induced interacting proteins of AC in living cells. Completion of the proposed studies will deliver mechanistic information regarding specific G protein subunits and new protein targets that could ultimately be used to prevent the development and expression of heterologous sensitization in vivo. PUBLIC HEALTH RELEVANCE: Understanding the molecular mechanisms responsible for D2 dopamine receptor-induced sensitization of adenylyl cyclase has implications in a variety of diseases including schizophrenia, Parkinson's disease, and drug abuse. Sensitization of adenylyl cyclase signaling occurs following persistent activation of several G?i/o-coupled receptors. Thus, the information discovered here is also relevant to many G protein-coupled receptors that are targets of drugs used to treat pain, depression, and Alzheimer's disease (e.g., opioid, serotonin, and muscarinic receptors).